Carlile M et al. (2009), Strand selective generation of endo-siRNAs from...

XB-ART-39238

Nucleic Acids Res 2009 Apr 01;377:2274-82. doi: 10.1093/nar/gkp088.

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Strand selective generation of endo-siRNAs from the Na/phosphate transporter gene Slc34a1 in murine tissues.

Carlile M , Swan D , Jackson K , Preston-Fayers K , Ballester B , Flicek P , Werner A .

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Natural antisense transcripts (NATs) are important regulators of gene expression. Recently, a link between antisense transcription and the formation of endo-siRNAs has emerged. We investigated the bi-directionally transcribed Na/phosphate cotransporter gene (Slc34a1) under the aspect of endo-siRNA processing. Mouse Slc34a1 produces an antisense transcript that represents an alternative splice product of the Pfn3 gene located downstream of Slc34a1. The antisense transcript is prominently found in testis and in kidney. Co-expression of in vitro synthesized sense/antisense transcripts in Xenopus oocytes indicated processing of the overlapping transcripts into endo-siRNAs in the nucleus. Truncation experiments revealed that an overlap of at least 29 base-pairs is required to induce processing. We detected endo-siRNAs in mouse tissues that co express Slc34a1 sense/antisense transcripts by northern blotting. The orientation of endo-siRNAs was tissue specific in mouse kidney and testis. In kidney where the Na/phosphate cotransporter fulfils its physiological function endo-siRNAs complementary to the NAT were detected, in testis both orientations were found. Considering the wide spread expression of NATs and the gene silencing potential of endo-siRNAs we hypothesized a genome-wide link between antisense transcription and monoallelic expression. Significant correlation between random imprinting and antisense transcription could indeed be established. Our findings suggest a novel, more general role for NATs in gene regulation.

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Species referenced: Xenopus
Genes referenced: actl6a cntn1 pfn1

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	Figure 1. Schematic representation of the Slc34a1 gene in vertebrates. (A) Phylogenetic conservation of the corresponding transcripts. The organization of the sense transcript (top) is conserved in all vertebrates with 13 exons and 12 introns. Antisense transcripts have been cloned from the three species flounder, zebrafish and mouse. The intron/exon structure of the different antisense transcripts differs significantly between organisms. Other splice forms may be expressed at a low level and have not been cloned. (B) The Slc34a1 locus as it is displayed in Contig view in the public ensemble database. The Affymetrix probe sets recognize the 3′ end of the Slc34a1 transcript (1423279_at), a region downstream of Slc34a1 partly complementary to Pfn3 (1427627_at) and the Pfn3 transcript itself (1453962_at).
	Figure 2. Analysis of the mouse Slc34a1 antisense transcript. (A) Putative splice products were tested by RT–PCR using testis total RNA. Only primers located within sense exon 10 and at the profilin 5′end (f1-r1; f2-r1) gave detectable amplicons. The 2 kb band (f3-r1) derived from genomic DNA. If genomic DNA was used instead of cDNA long range PCR yielded the expected fragments of 2 kb (f3-r2) and about 6.5 kb (f1-r2 and f2-r2, data not shown). (B) Tissue distribution of the antisense transcript in mouse testis, kidney and skeletal muscle. The lower panels represent the negative control minus reverse transcriptase and β-actin, respectively. The locations of the primers are indicated on the scheme in (A).
	Figure 3. Expression of sense and antisense transcripts in Xenopus oocytes. (A) Injection of the different constructs into the cytoplasm (C) or nucleus (N) of oocytes. All the cRNAs remain stable regardless of cytoplasmic or nuclear injection. (B) Co-injection of sense and various truncated antisense constructs. The size of overlap is indicated. A 30 bp overlap is efficiently processed whereas a 29 bp overlap is relatively stable.
	Figure 4. Northern blot analysis of short RNAs from injected oocytes and mouse tissues. The left panels (lanes 1–8) show samples from mouse, the right panel control samples from zebrafish (lanes 9–11). Lanes 1–3 indicates sense-antisense processing whereas lanes 4 and 5 do not contain short RNAs either because the overlap is too short (lane 4) or the samples were injected into the cytoplasm (lane 5). Lanes 6–8 represent tissues that express the sense encoded protein (kidney), do not express the transporter (skeletal muscle) or expresses both sense and antisense RNAs but the presence of the transporter is unclear (testis). Lane 10 represents short RNAs isolated from 48 h zebrafish embryos (19) as a positive control. Lane 11 shows another control, i.e. a sample from Xenopus oocytes injected into the nucleus with sense and antisense RNA. ‘Sense’- and ‘antisense’- probes mean that the short RNAs detected with the sense probe will be complementary to the antisense transcript and vice versa.
	Figure 5. Model for the biological role of antisense transcripts. (A) The ‘normal’ case where the antisense driving promoter becomes silenced. (B) The situation of a mutated allele (red star). The discontinuous nature of transcription may influence the timing of promoter silencing. This could ensure that the feedback mechanism remains allele specific.

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